Trichosanthin (TCS) is a plant protein which is obtained from the Trichosanthes kirilowii root tuber. The protein, which is also known as alpha-trichosanthin (Law) and Radix trichosanthis (Kuo Fen), is a basic, single-chain protein having a molecular weight of about 25,000 daltons. An incorrect protein sequence of TCS has been reported (Gu; Wang), and a molecular model has been derived from X-ray analysis (Pan).
It has been shown that TCS is a potent inhibitor of protein synthesis in a Cell-free lysate system (Maraganore). This activity is consistent with the observed homology in amino acid sequence between TCS and the A chain of ricin, a ribosome-inactivating protein (RIP) which shows amino acid homology with a number of other RIPs, including abrin A chain (Olnes, 1982, 1987) and modeccin (Olsnes, 1982), and various single chain ribosome-inactivating proteins, such as pokeweed anti-viral protein (PAP) (Irvin), RIPs from a variety of other plants (Coleman; Grasso; Gasperi-Campani) and the A subunit of Shiga-like toxins from E. Coli (Calderwood).
TCS, or plant extracts containing TCS, have been used in China as an abortifacient agent in humans, particularly during midtrimester (14 to 26 weeks). As such, the drug has been administered by intramuscular, intravenous, or intraamniotic routes, typically at a single dose of between about 5-12 mg. The phenomenon of mid-term abortion has been attributed to the selective destruction of placental villi. Other studies indicate that the syncytiotrophoblast is preferentially affected (Hsu; Kao) and that secretion of hCG may be impaired (Xiong). TCS has also been shown to have a suppressive effect on human choriocarcinoma, and the protein appears to be able to pass the blood/brain barrier (Hwang).
It has recently been shown that TCS has a selective inhibitory effect on viral expression in human T cells and macrophages infected with human immunodeficiency virus (HIV). This is evidenced by nearly complete inhibition of HIV-derived antigen in infected cells treated with the protein, as well as selective inhibition of protein and DNA synthesis in the infected cells. Similar results were also discovered for momorcharin, a basic glycoprotein obtained from the seeds of the bitter melon plant (Falosia; Spreafico; Lin; Barbieri). These findings, and applications of the two proteins for the treatment of HIV infection, are detailed in U.S. Pat. No. 4,795,739 for "Method of Selectively Inhibiting HIV".
Particularly in view of the ability of TCS to inhibit viral expression in HIV-infected human T cells and macrophages, it would be desirable to produce a relatively pure, invariant preparation of TCS, for use as a human therapeutic agent. Methods of preparing TCS from the roots of T. kirilowii have been reported (Yueng). Analysis of the purified TCS produced by earlier disclosed known methods indicates that the protein is only partially purified, and in particular, contains hemagglutinating contaminant protein(s). A more recent purification method described in co-owned patent application for "Purified Trichosanthin and Method of Purification", U.S. application No. 07/333,181, filed 4 Apr. 1989, yields a highly purified TCS preparation which is substantially free of protein contaminants, including hemagglutinating proteins.
Additionally, it would be desirable to produce TCS by means of recombinant DNA technology. Synthesis of the protein by recombinant methods would avoid the difficulty of obtaining T. kirilowii roots in fresh form, since at present the tuber roots are available only from Certain regions of the Orient. Recombinant production of TCS would also avoid the problem of variations in primary amino acid sequence in TCS obtained from natural root material from different geographic areas.
Recombinant production of TCS would also facilitate the production of peptide derivatives of TCS, including bioactive peptide portions of TCS, and bioactive portions of the protein fused with functional peptides which confer, for example, enhanced targetcell specificity.